Absolute (molar) quantification of proteins provides the analytical rationale for system-level modelling of diverse molecular mechanisms. FastCAT method employs multiple short (<50 kDa) stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q-)peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R-)peptides that relate its abundance to a single protein standard (BSA). FastCAT not only alleviates the need in purifying CP or using SDS-PAGE, but also improves the accuracy, precision and dynamic range of the absolute quantifications by grouping Q-peptides according to the expected abundance of target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantifications of neurological markers in human cerebrospinal fluid at the low ng/mL level.

LC-MS/MS was performed on a Q Exactive HF (Thermo Scientific, Germany) hybrid tandem mass spectrometer coupled with Eksigent 400 nanoLC system (Sciex, Germany) using the Nanospray Flex source (Thermo Fisher Scientific, Germany). Protein digests were loaded onto a trap column for 5 min at 7 µL/min and separated on the Acclaim PepMap 100 column (C18, 3 µm, 75 um x 150 mm) using 120 min gradients (5-45% B) at 300 nL/min in parallel reaction monitoring (PRM) mode. Scheduled PRM method consisted of MS1 scan from m/z 350 to 1700 with automatic gain control (AGC) target value of 3×106; maximum injection time (IT) of 60 ms and targeted mass resolution R (m/z=200) of 60,000. MS/MS was acquired with an inclusion list of 99 precursors; precursor isolation window of 1.6 Th; AGC of 1×106; a maximum IT of 80 ms; Rm/z=200 of 30,000 and NCE of 25%.

Aliquots of cerebrospinal fluid (CSF) of 20 µL volume were in-solution digested with trypsin/Lys-C protease mix (1:20) in the presence of RapiGest (Waters) detergent as described elswhere. CSF samples were obtained from patients diagnosed with relapsing-remitting multiple sclerosis and stored as freshly frozen aliquots. All patients gave their prior written consent. The study was approved by the institutional review board of the University Hospital Dresden.